This invention relates to the field of microbiology, and in particular to antimicrobial compositions, particularly to antimicrobial compositions that yield polymeric films, coatings, or shaped articles having prolonged antimicrobial activity, particularly in both the light and the dark.
The potential for the presence of pathogenic bacteria and viruses in biological fluids such as saliva, tears, blood, and lymph is of significant concern as is the potential for the transfer of such microorganisms to the surfaces of medical devices (and vice versa). For these reasons, methods for minimizing the transmission of pathogens in the home and in hospitals, as well no as in daycare centers, are important.
Microorganisms (e.g., viruses, bacteria, fungi) can be killed or rendered static by a number of physical and chemical methods. Physical methods include heat and radiation. There are a number of chemicals that have been used to limit viral, fungal, and bacterial growth. Examples include alcohols (usually as 70% by volume aqueous ethyl- or isopropyl alcohol), phenol (carbolic acid) and phenol derivatives such as hexachlorophene, formaldehyde, glutaraldehyde, ethylene oxide, ether, detergents, chlorhexidine gluconate, heavy metals such as silver, copper, and mercury, organic compounds of mercury such as mercurochrome, oxidizing agents such as hydrogen peroxide, iodine, hypochlorite, and chlorine. A number of antiviral agents are also known, including amantadine, nucleoside analogs such as AZT, aciclovir, ganciclovir, and vidarabine.
Antibiotics, such as bacitracin, the cephalosporins, cycloserine, the penicillins, vancomycin, chloramphenicol, the erythromycins, the tetracyclines, the sulfonamides, and the aminoglycosides (such as streptomycin, neomycin, and gentamycin), have traditionally been defined as chemicals made by microorganisms that kill bacteria. Antibiotics have no effect on viruses.
Such treatment methods are neither permanent nor continuous. thus repeated treatments may be needed. Compositions intended for imparting a continuously antimicrobial, self-disinfectinig property to surfaces or liquids have been disclosed, most of which involve covalent attachment of an antimicrobial moiety to a polymer or mixture of an antimicrobial agent with a polymer to impart controlled release of the antimicrobial agent.
Generally, known compositions intended for imparting continuous antimicrobial, self-disinfecting activity require intimate contact of the antimicrobial agent or antimicrobial moiety with a given bacterium, fungus, or virus. Since surfaces, in particular, inevitably become soiled, potentially precluding intimate contact of an antimicrobial agent or moiety with the contaminating microbe, it would be of potential benefit to have a method for imparting continuous antimicrobial, self-disinfecting activity at-a-distance,
Such a method was disclosed by Dahl et al., Photochemistry and Photobiology, 46, 3, 345-352 (1987) in which E. coli were separated from a surface by about 0.65 mm, wherein the surface included rose bengal. The method involved irradiating the rose bengal using visible light. The antimicrobial activity at-a-distance was ascribed to the diffusion of toxic singlet oxygen through air to the bacteria. Singlet oxygen itself is known to be generated by irradiation of rose bengal and other so-called triplet sensitizers.
Singlet oxygen is generated in neutrophils and macrophages for use in killing microorganisms. Superoxide dismutases, catalases, and peroxidases are defenses against radical- and reduced-oxygen species, but are not effective against singlet oxygen. A few microorganisms, such as Cercospora, are inherently resistant to singlet oxygen, and Gram-positive bacteria are generally more easily killed by singlet oxygen than Gram-negative bacteria. Enveloped viruses are inactivated by singlet oxygen more readily than nonenveloped viruses. It is notable that not a single documented case of acquired resistance by a bacterium, fungus, or virus to singlet oxygen is known.
The xe2x80x9cphotodynamic effectxe2x80x9d is the term used to describe destruction of cells and microbes by triplet-sensitizers in the presence of light. Under conditions where oxygen concentration is high and there are no reducing agents present, singlet oxygen is believed to be the destructive agent. This is the predominant mechanism (the so-called Type II mechanism) for cell destruction in cases where the photosensitizer cannot enter the cell. The Type II mechanism is known to be the predominant means of phototoxicity to E. coli for the xanthene dyes, such as rose bengal, for example, which upon irradiation generates reactive oxygen species. 80% of which are singlet oxygen, and 20% of which are superoxide radical anions. For photosensitizers that can pass through the lipid bilayer membrane into the interior of the cell where reducing agent concentrations, such as NADPH and glutathione, are high, the so-called Type I mechanism has been determined to be the predominant one leading to cell destruction. This mechanism involves, ultimately, the formation of a photosensitizer free radical and hydrogen peroxide, hydroxyl radical, and superoxide radical anion.
Some effort has been directed toward utilization of a combination of light and triplet-sensitizers (e.g., phthalocyanine, porphyrin hypericin, and rose bengal) for killing bacteria and fungi and for inactivating viruses. For example, photoinactivation of influenza virus by rose bengal and light was disclosed by Lenard et al., Photochemistry and Photobiology, 58, 527-531 (1993). Also, International Patent Application No. WO 94/02022 discloses improved germicidal compositions utilizing rose bengal in photodynamic killing of microorganisms on surfaces.
As stated above, chemical attachment (e.g., covalent or ionic) of photosensitizers to, or physical mixing of photosensitizers with, polymers has been of significant interest to workers in this field. Incorporation of dyes, such as xanthene dyes like rose bengal, into polymer matrices has been described in U.S. Pat. No. 5,830,526 (Wilson et al.), for example, which describes a woven or nonwoven fabric bound with a non-leachable light-activated dye by a cationic or anionic binder such as a water soluble polymer or carrageenan. Upon exposure to normal light, the dye generates singlet oxygen that kills microorganisms and viruses. As shown in Example 4 of U.S. Pat. No. 5,820,526, no dark antimicrobial activity is observed for the compositions comprising binder, and as Comparative Example 1 shows (below), when no binder is used, the dyes leach from the substrate to such a great extent that the compositions colorize articles with which they come in contact. Japanese Patent Application No. 5-39004 discloses ionic bonding of rose bengal to a positively charged polymer carrier and killing of microbes in the presence of oxygen and light. Bezman et al. Photochemistry and Photobiology, 28, 325-329 (1978) disclose the photodynamic inactivation of E. coli by rose bengal immobilized on polystyrene beads. It is believed that none of these examples of polymer-bound photosensitizers such as rose bengal, however, would have antimicrobial activity in the dark.
Generally, triplet-sensitizing dye compositions intended for imparting continuous antimicrobial, self-disinfecting activity utilize the dye in combination with light, thus severely limiting applications of these compositions to those where irradiation is feasible. Thus, as an example, a floor finish comprising one of the photodynamic compositions discussed above could impart antimicrobial activity to a floor during the day, or while the flooring is otherwise irradiated with visible light, but would not impart antimicrobial activity to the flooring during dark periods. Some dyes, however, such as methylene blue and halogenated xanthene dyes such as rose bengal, possess light-independent (dark) cytotoxic activity, and thus are effective antimicrobial agents in the dark as well as in the light. See, for example, Smith et al., Soil. Sci., 58, 47 (1944), Heitz et al., Light-Activated Pesticides, ACS Symp. Ser. 339, 1-21 (1987), and Scheffer et al., Investigative Ophthalmology and Visual Science, 35 3295-3307 (1994).
While the mechanism of the dark microbicidal activity of photosensitizers is unknown, it is clear that intimate contact of the photosensitizer with the microorganism is necessary. This is in contrast to the Type II mechanism of microbicidal activity of photosensitizers in the light, for which intimate contact of the photosensitizer and the microorganism is not necessary, due to the involvement of diffusible singlet oxygen. Heretofore, compositions allowing intimate contact of photosensitizer and microbe, which necessarily requires either direct application of photosensitizer in solvent (aqueous or organic), or leaching of photosensitizer from the compositions, have been avoided due to discoloration of skin and articles that come in direct physical contact with the leaching dye.
The present invention provides compositions that can be used to coat a wide variety of surfaces or form a wide variety of self-supporting polymer films or articles of a variety of shapes. The compositions include one or more polymers and one or more photosensitizers. Once a desired article (e.g., film) or coating is formed from the compositions, they are allowed to harden (e.g. cure) to form hardened polymer compositions. These hardened polymer compositions possess antimicrobial activity, preferably in the light as well as in the dark. Furthermore, they preferably do not visually colorize (i.e., discolor) skin or articles that come in contact with the resulting hardened polymer composition.
Thus, in one embodiment of the present invention, a method of limiting the presence of a microorganism is provided. The method involves contacting the microorganism with a hardened polymer composition comprising one or more polymers and one or more photosensitizers (preferably, a xanthene photosensitizer) wherein the polymer composition possesses antimicrobial activity (i.e., is capable of limiting the presence of a microorganism) in the light (e.g., room light) and the dark (i.e., the substantial absence of light). In another embodiment, an article is provided that includes a hardened polymer composition comprising one or more polymers and one or more photosensitizers wherein the polymer composition possesses antimicrobial activity in the light and the dark.
Significantly, such hardened polymer compositions and the articles that incorporate them can be produced according to the teachings of the present invention so as to not visually colorize skin or articles that contact them during use, due to the photosensitizer present in the hardened polymer composition. Conditions of use will vary depending upon the article and its application. This will be apparent to one of skill in the art.
Alternatively, such hardened polymer compositions and the articles that incorporate them can be produced according to the teachings of the present invention, which do not visually colorize, due to the photosensitizer, a piece of 95% by volume ethanol/5% by volume water solution-saturated white test paper placed in contact with the hardened polymer composition under 50-grams/cm2 pressure for 5 minutes. Preferably, the xcex94E value using a control portion of the test paper and a portion contacted with the hardened polymer composition is no greater than about 2.0
Preferably, at least one of the photosensitizers in the hardened polymer compositions (and the articles incorporating them) has the following formula: 
wherein the negative electric charges are balanced independently with the cations Na+, K+, Li+, H+, or substituted ammonium; each A independently represents hydrogen, chlorine, bromine, or iodine; and each B independently represents hydrogen, chlorine, bromine, or iodine. Preferred examples of such photosensitizers include those selected from the group of rose bengal, erythrosin, eosin yellowish, fluorescein, and mixtures thereof.
A hardened polymer composition of the present invention can be in the form of a coating, self-supporting film, or shaped article, for example. It can form a part of a surgical drape, a surgical face mask, pre-surgical patient prep, IV prep, handwash, dental appliance or other dental equipment, cosmetic applicator, sponge, contact lens, contact lens case, catheter (e.g., IV and urinary catheter), hospital gown, surgical glove, stethoscope, or equipment cover such as a keyboard cover or light switch cover. In addition, outdoor surfaces may also incorporate the photosensitizers of the present invention. In particular outdoor surfaces where microorganism growth can be a problem and would benefit by incorporation of the photosensitizer compositions of the present invention include roofing materials such as shingles, wooden shakes, tiles, and the like; cement and cement block; paints and stains for wood and other surfaces; road signs and the like.
Thus, the present invention provides an article comprising a hardened polymer composition comprising one or more polymers and one or more photosensitizers, at least one of which is a xanthene photosensitizer, wherein the hardened polymer composition posseses antimicrobial activity in the light and the dark and does not visually colorize white test paper saturated with a 95% by volume ethaniol/5% by volume water solution and placed in contact with the hardened polymer composition comprising the one or more photosensitizers under 50-grams/cm2 pressure for 5 minutes
A particularly preferred article is a contact lens case comprising a hardened polymer composition comprising one or more polymers and one or more photosensitizers wherein the hardened polymer composition possesses antimicrobial activity. Another article is a stethoscope comprising a hardened polymer composition comprising one or more polymers and one or more photosensitizers wherein the hardened polymer composition possesses antimicrobial activity. Preferably, the hardened polymer composition possesses antimicrobial activity in the light and the dark and does not visually colorize white test paper saturated with a 95% by volume ethanol/5% by volume water solution and placed in contact with the hardened polymer composition comprising the one or more photosensitizers under 50-grams/cm2 pressure for 5 minutes.
The present invention also provides methods of providing an antimicrobial surface, the method comprising combining one or more polymers with one or more photosensitizers to form a surface comprising a hardened polymer composition comprising one or more polymers and one or more photosensitizers. In one embodiment the photosensitizers are preferably xanthine photosensitizers, which are used in an amount such that the hardened polymer composition possesses antimicrobial activity in the light and the dark and does not visually colorize white test paper saturated with a 95% by volume ethanol/5% by volume water solution and placed in contact with the hardened polymer composition comprising the one or more photosensitizers under 50-grams/cm2 pressure for 5 minutes. Preferably the polymer is a non-cellulosic polymer and the photosensitizers are not bound to the polymer through covalent interactions. The non-cellulosic polymer is preferably a non-addition polymer.
For the purposes of this invention, the terms xe2x80x9climiting the presence of a microorganismxe2x80x9d or xe2x80x9cantimicrobial activityxe2x80x9d includes limiting the presence of at least one virus, at least one bacterium, at least one fungus, or a combination thereof. Limiting the presents of microrganism includes limiting the growth of a microorganism. This term also includes inhibiting, inactivating, killing, or preventing the replication of or reducing the number of a microorganism. Different terms may be used for different microorganisms.
The terms xe2x80x9climiting the presence of a virus,xe2x80x9d xe2x80x9cinactivation of virus,xe2x80x9d and xe2x80x9cviricidal activityxe2x80x9d as used herein refer to a reduction in the amount of virus present in a sample contacted with the hardened polymer composition of this invention. Preferably, the terms refer to an at least about 50% reduction in the amount of at least one species of virus detected on a surface of the hardened polymer composition relative to the same hardened polymer without the one or to more photosensitizers under the same conditions, using the test method as described in Example 5 below. More preferably, the compositions of the present invention provide at least about 75% reduction in the amount of at least one species of virus, even more preferably, at least about 90% reduction, and most preferably, at least about 99% reduction in at least one species of virus.
The term xe2x80x9climiting the presence of a fungus or a bacteriumxe2x80x9d as used herein refers to methods that employ the use of hardened polymer compositions described in this invention to inhibit, kill, or prevent the replication of or reduce the number of bacteria or fungi present on a surface of the hardened polymer composition. Preferably, the term refers to an at least about 40% reduction (as evidenced by the inhibition of growth or killing, for example) in the amount of at least one species of fungus or bacterium detected on a surface of the hardened polymer composition relative to the same hardened polymer without the one or more photosensitizers under the same conditions, using the test method described in Example 6 below. For example, growth of bacteria or fungi is limited by the polymer compositions of this invention when disks cut from the hardened polymer composition preferably kill at least about 40% or more of the bacteria or fungi placed on them, in the light as well as in the dark, as evidenced by washing away the original bacteria or fungi, attempting to grow colonies on an agar surface, and observing a reduction in the number of colonies that grow in comparison to the original inoculum and a control that does not include one or more photosensitizers of the present invention. More preferably the compositions of the present invention provide at least about 75% reduction, even more preferably, at least about 90% reduction, and most preferably, at least about 99% reduction in the amount of at least one species of fungus or bacterium detected on a surface of the hardened polymer composition relative to the same hardened polymer without the one or more photosensitizers under the same conditions, using the test method described in Example 6 below.
The term xe2x80x9ccontactingxe2x80x9d as used in the methods of this invention includes either physical contact of the polymer compositions of this invention with a virus, a bacterium, or a fungus, or exposure without direct physical contact of a virus, a bacterium, or a fungus to the hardened polymer compositions of this invention. Without intending to limit the scope of this invention, many of the photosensitizers of this invention may form diffusible substances in the light, such as singlet oxygen, which mediate an antimicrobial effect on the virus, bacterium, or fungus. Therefore, direct physical contact may not be necessary.
The term xe2x80x9cbacteriostaticxe2x80x9d refers herein to the property of inhibiting bacterial growth but not necessarily killing the bacteria. The term xe2x80x9cbactericidalxe2x80x9d refers to killing bacteria. The term xe2x80x9cfungistaticxe2x80x9d refers to the inhibition of replication of a fungus while the term xe2x80x9cfunticidalxe2x80x9d refers to killing the fungus. Thus, the polymer compositions of this invention can be either bactericidal or bacteriostatic or fungicidal or fungistatic. Methods for limiting the presence of a bacterium and fungus include xe2x80x9ccidalxe2x80x9d (i.e., killing) activity.
The language xe2x80x9cdoes not visually colorize skin or articlesxe2x80x9d as used herein means that contact of the hardened polymer composition with skin or other surface of an article does not cause the color of the skin or article to visually change color during customary use due to the photosensitizer. This does not necessarily mean that the polymer composition of the present invention is not colored itself, rather, it means that the hardened polymer composition does not transfer a significant amount of photosensitizer to skin or another article. The amount of coloration (i.e., transferred color) can be reported as a xcex94E (delta E) value. xe2x80x9cxcex94Exe2x80x9d is calculated according to the CIE 1976 Color Difference Formula, used for determining the color difference of two materials, such that xcex94E=[(L1xe2x88x92L2)2+(a*1xe2x88x92a*2)2+(b*1xe2x88x92b*2)]xc2xd, wherein L1xe2x88x92L2 is the lightness difference between the two materials, a*1xe2x88x92a*2 is the difference in red-green light response of the two materials, and b*1xe2x88x92b*2 is the difference in blue-yellow light response of the two materials, which can be measured using various commercially-available instruments such as, for example, the Color-Difference Meter of Hunter as described by Judd and Wyszecki in Color in Business, Science, and Industry, second edition, published by John Wiley and Sons, Inc., New York, pages 295-296.
An xe2x80x9ceffective amountxe2x80x9d of one or more of the photosensitizers of this invention refers to an amount of the photosensitizer, as a weight percentage of the dry polymer weight, that is sufficient to limit the presence of at least one of a virus, a bacterium, or a fungus.
A xe2x80x9chardened polymer compositionxe2x80x9d is a combination of at least one hardened polymer and at least one photosensitizer. A xe2x80x9chardened polymerxe2x80x9dcan be achieved by solidifying a liquid polymer, crosslinking or otherwise curing a polymer to render it insoluble, by extruding or molding a polymer, etc.
It does not necessarily mean that the polymer is hard and inflexible; rather it means that the polymer is cured or otherwise rendered solid. In fact, in certain applications such as coatings on flexible or deformable substrates a flexible xe2x80x9chardenedxe2x80x9d polymer composition may be preferred. Furthermore, depending on the type of polymer, a xe2x80x9chardenedxe2x80x9d polymer may have been cooled and solidified (as for a thermoplastic) or cured (i.e., polymerized and/or crosslinked) from polymer precursors.